Stroke is a major cause of morbidity in the United States, affecting over 600,000 people annually. The only therapeutics currently available for the treatment of stroke are thrombolytic agents such as tPA, which have limited efficacy and are inappropriate for many patients due to risk of hemorrhage. One promising alternative to thrombolytics has been a class of agents that block glutamate-mediated excitotoxicity by targeting the NMDA receptor. These drugs have worked well in animal models of stroke but have failed in clinical trials, most often due to the appearance of neurological or cardiovascular side effects at dosages lower than the efficacious range. In a Phase I research program, Arbor Vita has identified a class of inhibitors that act downstream of NMDA receptors termed PSD-95 antagonists. These inhibitors are effective in rodent models of focal ischemia without blocking the electrical or calcium channel functions of the NMDA receptor. Preclinical development of the lead PSD-95 inhibitor, the fusion peptide TatNR2B9c, demonstrated impressive central nervous system safety, an important benefit versus traditional NMDA receptor blockers. Moreover, the long term toxicity of TatNR2B9c was minimal. Acute cardiovascular side effects were present, however, at doses only slightly higher than those predicted to be effective in humans. These effects were caused by a portion of the fusion peptide required for cellular uptake, but not involved directly in PSD-95 blockade. The focus of this proposal is to modify this portion of the fusion peptide, to overcome the cardiovascular side effects without a loss in potency or efficacy. The resulting improved PSD- 95 inhibitor would then be developed by carrying out essential preclinical safety and efficacy studies leading towards subsequent clinical testing and eventual commercialization. The Specific Aims of the proposal are: 1) identify TatNR2B9c derivatives that are cell permeable and bind PSD-95 potently, but do not trigger cardiovascular side effects, 2) demonstrate efficacy of the most promising drugs in the rat permanent pial vessel occlusion (P3VO) model of stroke, 3) confirm the improved safety of the lead peptides by conducting a dog cardiovascular (CV) safety study, 4) characterize the neuroprotective efficacy of the lead peptides as guided by the STAIR (Stroke Academic Industry Roundtable) recommendations, and 5) conduct the complete battery of preclinical safety and toxicity studies required to file a successful Investigational New Drug Application with the U.S. Food and Drug Administration. Completion of this research will lead to clinical development of a peptide therapeutic with the potential to revolutionize the treatment of stroke and thereby address a massive unmet medical need. PUBLIC HEALTH RELEVANCE: There are no approved drugs directed at protecting against the brain damage caused by stroke, only drugs which can help restore blood flow to the afflicted area. The present grant aims to improve and advance towards clinical testing drugs directed at a novel target identified by Arbor Vita that can be administered after a stroke occurs and greatly reduce the resulting brain damage.